CONCLUSIONES FINALES

The Src homology domains 2 (SH2) and 3 (SH3) were among the first to be characterised in signal transduction proteins. These SH domains were identified as regions outside the catalytic kinase domain (SHI domain) o f cytoplasmic protein tyrosine kinases (PTKs) which shared homology with other known signal transduction proteins (Mayer et ak, 1988; Sadowski et al., 1986).

SH2 domains are sequences o f about 100 amino acids and have been found in many proteins o f the signal transduction pathways which regulate phospholipid metabolism, tyrosine phosphorylation and dephosphorylation, activation o f Ras- family GTPases, gene expression, protein trafficking and cell structure (Pawson and Schlessinger, 1993). In üw, signal transduction proteins containing SH2 domains bind to the phosphotyrosine (pY) motifs both o f activated cell-surface receptors and cytoplasmic proteins (Anderson et ak, 1990; Matsuda et al., 1990; Valius and Kazlauskas, 1993). The pY-SH2 domain interactions can be reproduced in vitro using isolated SH2 domains and short pY-containing peptide sequences o f 5 - 10 amino acids. SH2 domains bind their optimal target sequences with high affinity, where the equilibrium dissociation constant, Kg=10 - 100 nM. Peptides containing random sequences around the pY residue bind SH2 domains around 1000-fold less strongly. SH2 domains have essentially no affinity for non-phosphorylated peptides. Thus, the strength o f binding comes mostly from the pY-SH2 domain interaction, while the surrounding amino acids contribute to the fine specificity o f binding (Piccione et al., 1993).

Many structural studies have shown that SH2 domains exhibit a well- conserved 3-D topology comprising a central (3-sheet sandwiched between two a- helices (e.g. (Waksman et al., 1992)) (Figure 1-S), The structurally characterised SH2 domains all bind pY residues in the same way, i.e. in a specific pocket o f the SH2 domain that contains a strictly conserved arginine residue. The conserved arginine hydrogen-bonds with two pY oxygen atoms, while the loop between (3-strands B and C closes over the phosphate (e.g. (Lee et al., 1994)). In addition to the pY binding pocket, there is a groove on the SH2 domain surface containing variable amino acids which confer the specific recognition o f the three residues to the C-terminus o f the pY residue in the target ligand (denoted +1 to +3) (Eck et ak, 1993; Waksman et ak, 1993). Two types o f consensus m otif in the +1 to +3 region have been identified: the

sequence o f type pY-hydrophilic+^-hydrophilic+^-I/P+a binds to a group o f SH2 domains which includes those from Src, Fyn, Lck and Nek; while the SH2 domains o f p 8 5 a /p ll0 a PI3-kinase and PLC-y prefer a sequence o f the t)'pe pY- hydrophobic+i-X+2-hydrophobic+3, where X is any amino acid (Songyang et ak, 1993;

Zhou et ak, 1993).

It appears that the bipartite nature o f the pY-SH2 domain interaction allows the tyrosine phosphorydation mechanism to act as an all-or-nothing signalling switch. I'his interaction does not seem to have major allosteric effects and therefore probably represents a simple device for the recruitment o f signal transduction proteins (Sabe et ak, 1994). Since this recruitment is frequently performed by a RTK, it often results in the co-ordinated phosphorydation o f the recruited signal transduction protein (Rotin et ak, 1992).

¥ignn 1-5: The backbone and secondary structure etemenfs of the z;-Src S I 12 domain (W'aksman et al., 1992). The consened S H 2 domain topology is clearly seen, i.e. an anti-parallel three-stranded P~sheet flanked by two a-helices. This diagram type was made using MOLSCRIPT (Kraulis, 1991).

O A

« B

Like SH2 domains, SH3 domains are found in many signal transduction proteins, but also in proteins o f the cytoskeleton (Drubin et ak, 1990; Koch et ak,

1991). SH3 domains are composed o f 55 - 70 amino acids which fold into a compact domain o f five (3-strands organised into two roughly orthogonal (3-sheets {Figure 1-

(5) (structures have been determined both by X-ray cr^^stallography (Musacchio et ak, 1992) and NMR (Yu et ak, 1992)). SH3 domains appear to be important for mediating the constitutive or reversible protein-protein interactions o f many signal transduction proteins. For example, the two SkI3 domains o f the adapter protein GRI3-2 mediate its constitutive interaction with SOS (Egan et ak, 1993). Thus, the signal-dependent recruitment o f GRB-2 to RTKs also recruits SOS — a guanine nucleotide exchange factor (GEF) and activator o f Ras (Bonfini et ak, 1992). In this way, RTK activation co-localises the GRB-2/SOS complex with membrane-bound Ras, leading to Ras activation and the consequent stimulation o f the MAPK pathway. In addition, the Src-family and Btk/Tec-family kinases have SH3 domains which undergo regulated interactions with the proline-rich sequences o f different signal transduction proteins (see below). The in mvoimportance o f SH3 domain interactions is further underlined by the demonstration that mutations in the Src STI3 domain result in increased catalytic activit)^ and oncogenic potential o f Src.

Figure 1-6: The 3-D stmctiire of the ^-spectrin SH 3 domain (hhisacchio et al., 1992). SH 3 domains Jom s compact stmctnres of five ^-strands organised into two rvnghlj orthogonal ^-sheets.

SH3 domains bind to proline-rich sequences o f about 10 amino acids, where for the isolated SH3 domain in vitro^ the Kd=5-100 |iM (Ren et al., 1993; Yu et al., 1994). In general, the specificity o f binding is conferred by interactions between the non-proline amino acids o f the ligand and the non-conserved amino acids in two charged loops which flank the hydrophobic binding pocket on the SH3 domain surface (Cohen et al., 1995). When bound to SH3 domains, proline-rich sequences usually adopt a left-handed (polyproline) type II (PPII) helix, which has three residues per turn (Feng et al., 1994; Lim et al., 1994; Musacchio et al., 1994). The PPII helix has three spines, two o f which form a roughly planar surface and contact the SH3 domain. The more exposed spine tends to contain proline residues which probably serve to stabilise the PPII helix structure. Although a consensus SH3 domain ligand was previously defined as X-p-hydrophobic-P-p-X-P (where X is any amino acid, p tends to be a proline residue and P is a conserved proline), it has recently been shown that a sequence containing only one proline residue is sufficient for PPII helix formation and intramolecular binding in the repressed, phosphorylated form o f the Src tyrosine kinase (Xu et al., 1997). Indeed, other recent studies have also revealed intramolecular SH3 domain-mediated interactions in Src-family kinases (Sicheri et al., 1997) and in Itk, a Btk/Tec-family kinase (Andreotti et al., 1997). It is o f great interest that some o f these intramolecular SH3 domain interactions exhibit kinase-regulating abilities which, moreover, can be modulated by other signal transduction proteins (Andreotti et al., 1997; Moarefi et al., 1997). Therefore, a clear role for the SH3 domains o f multiple signal transduction proteins has been demonstrated and it seems likely that SH3 domains may co-ordinate a whole array o f other signalling interactions yet undiscovered.